Topic 7: Sampling, Standardization and Calibration

Topic 7
Sampling, Standardization and

Calibration
1
2
A sample that represents the concentration of
various substances under study in the
manufactured products ?
3
A sample that provides useful information of
an incident ?
4
A sample that represents the concentration of
toxic chemicals of a waste dump site ?
5
Type of Samples and Methods
What is Qualitative & Quantitative analyses?
Qualitative analyses : To identify chemical species
Quantitative analyses : To determine the amount of a constituent
Quantitative methods :
-Traditionally, eg. Gravimetric , volumetric, and instrumental
To distinguish suitable analyses methods is based on the size of sample and

the level of constituent.
6
Classification of Modern Analytical Methods
 Gravimetric Methods determine the mass of the analyte or some compound

chemically related to it.
 Volumetric Methods measure the volume of a solution containing sufficient reagent

to react completely with the analyte.
 Electroanalytical Methods involve the measurement of such electrical properties as

voltage, current, resistance, and quantity of electrical charge.
 Spectroscopic Methods are based on the measurement of the interaction between

electromagnetic radiation and analyte atoms or molecules, or the production of such
radiation by analytes.
 Miscellaneous Methods include the measurement of such quantities as mass-to-

charge ratio, rate of radioactive decay, heat of reaction, rate of reaction, sample
thermal conductivity, optical activity, and refractive index.
7
Steps in a quantitative analysis:
Obtain a Prepare a Define

Select Method representative laboratory replicate
sample sample samples
Measure
Calculate Eliminate Dissolve the
property of
results interferences samples
the analyte
Estimate the
reliability of
“Typical Quantitative Analyses”
results
Reference: Skoog, C. D., West, D. M., & Holler, F. J., (1991). Fundamentals of Analytical Chemistry (7th
Edition), John Wiley & Sons, Hoboken , NJ.
8
Classification of analyses by sample size :
Macro
Type of Analyses
Semimicro
Micro
Submicro
Ultramicro
0.0001 0.001 0.01 0.1

Sample size, g
Example:
- Analysis of a 1g sample of soil for a suspected pollutant would be called a macro

analysis, while that of a 5mg sample of a powder suspected to be an illicit drug would
be a micro analysis.
Reference: Skoog, C. D., West, D. M., Holler, F. J., & Crouch, S. R. (2004). Fundamentals of Analytical Chemistry
(8th Edition), John Wiley & Sons, Hoboken , NJ.
9
Classification of constituent types by analyte level:
Type of Constituent Major
Minor
Trace
Ultratrace
1 ppb 1 ppm 0.1% 100%
Analyte Level
Examples:
- Determination of Hg in the ppb to ppm range in a 1-μL (≈ 1mg) sample of river water
would be a micro analysis of a trace constituent.
Reference: Skoog, C. D., West, D. M., Holler, F. J., & Crouch, S. R. (2004). Fundamentals of Analytical
Chemistry (8th Edition), John Wiley & Sons, Hoboken , NJ.
10
Select Method:
Examples of the bodies that have specified definite instructions for sampling given
materials:
International Organization for ASTM International

Standardization (ISO) (www.astm.org)
(www.iso.org)
AOAC International American Public Health Association

(www.aoac.org) (www.apha.org)
12
What is Sampling in Research??
13
Obtain a Representative Sample
- Involves obtaining a small mass of a material whose composition accurately

represents the bulk of materials being sampled.
- Steps in obtaining a laboratory sample:
Reduce the gross

Identify the Collect a gross sample to a
population sample laboratory sample
- The population refers the bulk materials being sampled.
-The items chosen for analysis are often called sampling units or sampling
increments.
- The collection of sampling units or increments are called gross samples.
- The gross sample is usually reduced in size and made homogeneous to become
the laboratory sample for analysis in the laboratory.
14
- Example: Determination of the average concentration of lead in the
collection of coins.
The collection of coins Five coins were collected
(say 1000 coins) (Each coin is known as a SAMPLING UNIT or
POPULATION SAMPLING INCREMENT)

The five coins collected are the GROSS
SAMPLE
The coins sample was reduced in size

(say a few grams or less) and made
homogenous (acid digestion etc.) for
analysis.
LABORATORY SAMPLE
15
Sampling
- Statistically, the goals of the sampling are:
i. To obtain a mean value that is an unbiased estimate of the population mean. This
goal can be realized only if all members of the population have an equal probability
of being included in the sample.
ii. To obtain a variance that is an unbiased estimate of the population variance so

that valid confidence limits can be found for the mean, and various hypothesis tests
can be applied. This goal can be reached only if every possible sample is equally
likely to be drawn.
- Both goals requires a random sample, a sample chosen by applying

randomization procedure . The sample can be randomly chosen by referring to a
table of random numbers. The random numbers can be conveniently generated
from a random number table or from a spreadsheet (MS Excel) using the random
number function [=RAND()].
16
Sampling
- Example: Random selection of a sample containing 100 horses from 380
horses using a Table of Random Numbers.
Taken from http://www.pitt.edu/~super1/lecture/lec0411/022.htm

17
Sampling
- Coning and Quartering: A process for reducing the size of gross sample.
Division of the flattened cone

Pouring an initial cone into halves
Removal of alternate quarters to define Further division of the flattened cone

the subdivided sample into quarters
H. G. Brittain. (2002). Pharmaceutical Technology, Page 67 18

Sampling Uncertainties
• All experimental uncertainty is due to either random errors or
systematic errors.
• Random errors are statistical fluctuations (in either direction) in the
measured data due to the precision limitations of the measurement
device. Examples: You measure the mass of a NaOH three times
using the same balance and get slightly different values: 7.64 g,
7.61 g, 7.68 g.
• Systematic errors, by contrast, are reproducible inaccuracies that
are consistently in the same direction. Examples: The electronic
scale you use reads 0.05 g too high for all your mass measurements
(because it is improperly tared throughout your experiment)
• Random errors, which are reflected in the precision of data, can
generally be kept at an acceptable level by close control of the
variables that influence the measurements.
• Systematic errors can be eliminated by exercising care, by
calibration, and by the proper use of standards, blanks, and
reference materials.
18
Sampling Uncertainties
- Sampling errors are ordinarily treated separately from other uncertainties associated with
an analysis.
- For random and independent uncertainties, the overall standard deviation soverall for an
analytical measurement is related to the standard deviation of the sampling process
ssampling and to the standard deviation of the method smethod by the relationship.
Soverall2 = Ssampling2 + Smethod2 Equation 1
- In many cases, the method variance will be known from replicate measurements of a
single laboratory sample. Under this circumstance, Ssampling can be computed from
measurement of Soverall for a series of laboratory samples, each of which is obtained from
several gross samples.
- When Smethod<Ssampling/3, there is no point in trying to improve the measurement precision.
The equation shows that Soverall is determined predominantly by the sampling uncertainty
under these conditions.
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The Gross Sample
- The gross sample is the collection of individual sampling units. It should correspond to the
bulk material in chemical composition and, if composed of particles, in particle size
distribution.
- Basically, the gross sample weight is determined by:

» The uncertainty that can be tolerated between the composition of the gross sample and
that of the whole.
» The degree of heterogeneity of the whole, and
» The level of particle size at which heterogeneity begins.
- To obtain a truly representative gross sample, a certain number N of particles must be

taken. The magnitude of this number depends on the uncertainty that can be tolerated
and the heterogeneity of the material. The number may range from a few particles to as
many as 1012 particles.
20
The Gross Sample
- Assuming there is a two-component mixture system containing two types of
particles with same size where type A particles containing the analyte type B.
The probability of drawing a type A particle is p. The number of particles N
needed to achieve a given relative standard deviation σr can be obtained from
the equation:
Equation 2
- Example: if 80% of the particles are type A (p = 0.8) and the desired relative
standard deviation is 1% (σr = 0.01), the number of particles making up the
gross sample can be calculated using the above equation.
21
The Gross Sample
- Equation 2 can be extended to a situation where both types of particles contain
the analyte in different percentages with the percentage of analyte in particle A,
PA is higher than type B, PB.
Equation 3
d = average density of particles (differs from the densities of particles A, dA

and particles B, dB
P = overall average percentage
σr = Sampling relative standard deviation
22
The Gross Sample
Question 1:
A column-packing materials for chromatography consists of a mixture of two
types of particles. Assume that the average in the batch being sampled is
approximately spherical with a radius of about 0.5 mm. Roughly 20% of the
particles appear to be pink and are known to have 30% by weight of a
polymeric stationary phase attached (analyte). The pink particles have a
density of 0.48 g/cm3. The remaining particles have a density of about
0.24g/cm3 and contain little or no polymeric stationary phase. What mass of
the material should be the gross sample contain if the sampling uncertainty is
to be kept below 0.5% relative?
23
The Gross Sample
24
The Gross Sample
25
The Gross Sample
- Equation 3 can be rearranged to calculate the relative standard deviation of
sampling, σr.
PA  PB d Ad B p(1  p)
r  P  2 Equation 4
d N
- The Ingamells sampling constant Ks is a sampling constant equal to the
mass of sample producing a percent relative standard deviation for sampling of
± 1%. It can be obtained from the following equation.
K S  mR  m  ( r 100%)
2 2
Equation 5
where m is the mass of the sample and R is the percent relative standard
deviation for sampling.
26
The Gross Sample
Question 2:
The following data were obtained in a preliminary determination of the
amount of inorganic ash in a breakfast cereal. Calculate the sampling
constant Ks for the samples.
Mass of cereal (g) Percentage ash (w/w)

0.9956 1.34
0.9981 1.29
1.0036 1.32
0.9994 1.26
1.0067 1.28
Reference: Harvey, D. (2000). Modern Analytical Chemistry. McGraw-Hill Publishing, Singapore.
27
The Gross Sample
28
The Gross Sample
29
The Gross Sample
Collections of various types of samples:
A) Homogeneous solutions of Liquids and Gases.

> Must be stirred well before sampling to ensure the homogeneity of
the samples.
> A “sample thief” can be used to collect the sample solution if the
volume of the solution is large (See figures for examples).
30
Examples of Sample Thief Sample Thief (Bacon Bomb)
Obtains samples from storage tanks, tank
cars and drums. When the thief strikes the
bottom of the tank, a plunger assembly
opens to admit the sample. The plunger
closes again when the bomb is withdrawn,
forming a tight seal. Samples can be taken
at any depth with the use of a secondary
trip line, or extension rods may be added
for obtaining samples at levels of up to 18
(46cm) off the bottom. Equipped with
plunger locking cam for tight closure during
transport (except for 4 oz 1 1/8" dia.
model). Special models include a 4 oz
(118mL) ‘pencil’ model for sampling
through small diameter pipes and
openings, and clear acrylic samplers with
plated brass plunger and end pieces.
31
Cage samplers can be
used to obtain:
Running Average
Sample, RVP Sample
and Spot Level
Sample
Weighted Beaker Sampler can be

used to obtain: All level sample
and Spot sample.
Zone Sampler can be

used to obtain: Spot
sample at any level
Stainless Steel & Brass Weights
The CS Restricted sampler shown can be used to obtain: RVP Sample,

All Level sample and Spot level sample. The restricted flow dial allows
user to adjust the amount of sample flowing into the sampler for
Examples of Sample
different viscosity products, which assist the user in obtaining a
accurate representative sample of all levels of the product.
Thief
Reference
LPG Hydrometer Cylinder
Part# 407B046 http://www.channelsupplies.com/sampling.htm
Conforms to ASTM D1657 &
GPA21-140 http://www.sampling.co.uk/ 32
The Gross Sample
Collections of various types of samples (continued):
A) Homogeneous solutions of Liquids and Gases (continued).
> On-site measurement (bring the laboratory to sample). However, it is
important to note that most portable sensors measure only the local concentration
and do not average or sense remotes concentrations.
A portable water testing equipment A portable Gas Chromatography-

Mass Spectrometer (GC-MS)
33
The Gross Sample
A) Homogeneous solutions of Liquids and Gases (continued).
> When samples of liquids are collected from flowing stream (river or flowing
gases in gas pipes for example), care must be taken so that the sample collected
represents a constant fraction of the total flow and that all portions of the stream
are sampled.
> Several methods of collecting gases samples:

1) Use a sampling bag.
2) Trap the gases in a liquid.
3) Absorb the gases onto the surface of a solid.
34
The Gross Sample
B) Particulate Solids.
> Steps in sampling a particular solid:
Randomly collect N Reduce particle size of

Identify the population to
(Eq 3) particles to gross sample and
be analyzed
give gross sample homogenize
No
Store the laboratory Is this sample of a Randomly collect N

sample Yes suitable size for the particles to give
laboratory ? laboratory sample
Remove portion of the laboratory

sample for analysis
(8th Edition), John Wiley & Sons, Hoboken , NJ. 35
The Gross Sample
B) Particulate Solids (continued).
> Random sampling of a bulky particulate material can best be
accomplished while the material is being transferred.
> Mechanical devices have been developed for handling many types
of particulate matter.
36
The Gross Sample
C) Metals and Alloys.
> The samples are obtained by sawing, milling and drilling.
> In general, it is not safe to assume that chips of metal removed from
the surface are representative of the entire bulk, so solid material
from interior must be sampled as well.
37
The Laboratory Sample
- For homogenous solids, the gross sample may weigh

from hundreds of grams to kilograms or more;
therefore reduction of the gross sample to a finely
ground and homogeneous laboratory sample,
weighing at most a few hundred grams, is necessary.
- The laboratory sample should have the same number

of particles as the gross sample.
- The process of preparation of a laboratory sample

involves a cycle of operations that includes crushing
and grinding, sieving, mixing and dividing the samples
(often into two halves) to reduce its weight.
38
- The number of laboratory samples N required to obtain a

tolerable relative uncertainty σr at a given confidence level
can be calculated from the following equation.
2 Equation 6
where t is the value obtained from the t-table at the given

confidence level.
39
Question 3:
A carload of lead ore containing galena (≈70% Pb) and other particles with little or
no lead is to be sampled. From the densities (galena = 7.6 g/cm3, other particles
= 3.5 g/cm3, average density = 3.7 g/cm3) and rough percentage of lead,
Equation 3 indicates that 8.45 x 105 particles are required to keep the sampling
error below 0.5% relative. The particles appear spherical with a radius of 5 mm.
A calculation of the weight required, similar to that in Question 1, shows that the
gross sample should weigh about 1.6 x 106 g(1.8 tons). If the gross sample has
to be reduced to a laboratory sample that weighs about 100g. How can this be
done ?
40
41
42
Question 4:
The determination of copper in a sea water sample gave a mean

value of 77.81μg/L and a standard deviation ss of 1.74μg/L.
(Note: The insignificant figures were retained here because
these results are used later in a calculation.) How many
samples must be analyzed to obtain a relative standard
deviation of 1.7% in the results at the 95% confidence level ?
43
44
Replicate Samples
Example: Determination of the average concentration of lead in the
collection of coins.
The collection of coins Five coins were collected

(say 1000 coins)
GROSS SAMPLE
POPULATION
The lead in the coins was extracted by

The diluted solution is divided into aqua regia (a mixture of HCl/HNO3) and
three equal portions (same volume) the solution was diluted for analysis.
and the lead content of three
portions of solution were measured LABORATORY SAMPLE
on the same day using the same
technique.
THREE REPLICATE SAMPLES
45
Replicate Samples
- Replicate samples are portions of a material of approximately the same size
that are carried through an analytical procedure at the same time and in the
same way.
- The purpose of performing chemical analyses on replicate samples is to

improve the quality of the results and provides a measure of their
reliability.
- For a situation where smethod2 is significantly greater than ssampling2, then only a
single sample need to be collected and analyzed. The number of replicate
analyses, Nr, needed to minimize the error due to the method is given by
Equation 7
where e2 is expressed as absolute uncertainties or as relative uncertainties.
References:
- Skoog, C. D., West, D. M., & Holler, F. J. (1996). Fundamentals of Analytical Chemistry (7th Edition), Saunders
College Publishing, Orlando, Florida.
46
- Harvey, D. (2000). Modern Analytical Chemistry. McGraw-Hill Publishing, Singapore.
Replicate Samples
- Unfortunately, the simple situations as shown in Equation 7 are often
exception. In many cases, both the sampling variance and method variance are
significant, and both multiple samples and replicate analyses of each sample
are required.
- The overall error for this circumstance is given by
Equation 8
where Ns is the number of samples.
- Equation 8 does not have a unique solution because different combinations of

Ns and Nr give the overall error. The choice of how many samples to collect and
how many times each sample should be analyzed is determined by other
concerns, such as the cost of collecting and analyzing samples, and the
amount of available.
References:
- Harvey, D. (2000). Modern Analytical Chemistry. McGraw-Hill Publishing, Singapore. 47
Replicate Samples
- Question 5:
A certain analytical method has a relative sampling variance

of 0.40% and a relative method variance of 0.070%. Evaluate
the relative error (α = 0.05) if (a) you collect five samples,
analyzing each twice; and, (b) you collect two samples,
analyzing each five times.
References:
Replicate Samples
- Solution for Question 5 (continued)
References:
Standardization and Calibration
- Calibration is a process of ensuring that the signals measured by a piece of
equipment or an instrument is correct.
- Standardization is the process of establishing the relationship between the

amount of analyte and a method’s signal.
- Analytes refers to the constituents of interest in a sample.
- Usually in a determination, the raw response is not used. Instead, the raw
analytical response is corrected by measuring a blank.
- A blank refers a solution that contains all the substances present in the
standards, and the unknown if possible, except for the analyte.
References:
- Skoog, C. D., West, D. M., Holler, F. J., & Crouch, S. R. (2004). Fundamentals of Analytical
- Kenkel, J. (2003). Analytical Chemistry for Technicians (3 Edition). CRC Press, Boca Raton. 50
- In practice, with complex samples, it is too time consuming or impossible to prepare an
ideal blank. Most often, a real blank is either a solvent blank containing the same
solvent in which the sample is dissolved, or a reagent blank, containing the solvent plus
all the reagents used in sample preparation.
- Usually the standardization process is accomplished by the use of chemical standards

(reagents of exactly known concentrations).
- A plot of signals of a series of standard solutions (Sstandard) versus the concentration of

the standards (Cstandard), known as the calibration curve is prepared by plotting the data
or by fitting them to a suitable mathematical equation.
- The most useful calibration curve is a straight line since the method’s sensitivity (the k
value in Equation 10) is the same for all concentrations of analyte.
References:
- Kenkel, J. (2003). Analytical Chemistry for Technicians (3 Edition). CRC Press, Boca Raton. 51
- If a straight-line relationship is assumed, then the data fit the equation for a straight line.
y = mx + b Equation 9
where y is the dependent variable, x is the independent variable, m is slope of

the curve, and b is the intercept on the ordinate(y axis). y is usually the
measured variable plotted as a function of changing x.
- A relationship between the measured signal, Smeasured and the relative amount of analyte
in a sample (Canalyte) is shown in the following equation.
Smeasured = kCanalyte + Sreagent Equation 10
where Canalyte is the analyte’s concentration, k is the method’ s sensitivity, and

Sreagent is the contribution to Smeasured from constant errors introduced by the
reagent used in the analysis. To obtain an accurate value for Canalyte it is
necessary to avoid determinate errors affecting Smeasured, k and Sreagent.
This is accomplished by a combination of calibrations, standardizations and
reagent blanks.
References:
- Christian, G. D. (2004). Analytical Chemistry (6th Edition). John Wiley & Sons, Hoboken, NJ. 52
53
- Ideally the reagents used should not give any error that might affect the
Smeasured, Measurement of a reagent blank corrects the error contributed by the
reagents used so that Sreagent become 0. The Smeasured is then mainly affected
by Canalyte and k. Equation 10 then become
Smeasured = kCanalyte Equation 11
References:
- Christian, G. D. (2004). Analytical Chemistry (6th Edition). John Wiley & Sons, Hoboken, NJ.
54
- Example: Blank Measurement for Correction of Raw Experimental Data
Concentration of Cu Signal Corrected signal

standard solutions (ppm) (Absorbance) (Corrected absorbance#)
0.0 0.002 0.000*

(blank)
1.0 0.078 0.076
2.0 0.163 0.161
4.0 0.297 0.295
6.0 0.464 0.462
8.0 0.600 0.598

* Zero absorbance is defined with a blank
# Corrected absorbance = (absorbance of sample) – (absorbance of blank)

55
- Example: Blank Measurement for Correction of Raw Experimental

Data (Continued)
> The signal of the reagent used to prepare the Cu standard solutions
(reagent blank solution), S reagent is measured.
> S reagent = y-intercept = 0.002, the correction is done by subtracting

all the readings with 0.002.
> The corrected reading for the blank solution is 0.000, the changes in
readings of the standard solutions can then be directly correlated
to the change of the concentrations of the analyte in the solutions if
the method’s sensitivity, k is kept constant, as shown in Equation 11.
56
- In practice, not all the data fall exactly on the line due to the indeterminate errors in the
measurement process.
- The investigator must try to draw the “best” straight line among the data points.
- Regression analysis (A mathematical technique for fitting an equation to experimental

data) provides the means for objectively obtaining such a line and also for specifying the
uncertainties associated with its subsequent use.
- The “best” straight line can be drawn using the method of least squares.
- In the method of least squares, the “best” straight line through a series of experimental
points is that line for which the sum of the squares of deviations (the residuals) of the
points from the line is minimum.
References:
- Christian, G. D. (2004). Analytical Chemistry (6th Edition). John Wiley & Sons, Hobken, NJ.
- Harris, D. C. (2010). Quantitative Chemical Analysis (8th Edition), W. H. Freeman and
Company, NY. 57
- Recall the key elements of a straight-line plot:
Equation : y = mx + b
yL = mxi + b
yL
Residual =yi – (mxi + b)
yi
∆y
Slope = ∆y/∆x = m = (y – b)/x
∆x
0 xi
y- intercept = b = y – mx
References:
- Christian, G. D. (2004). Analytical Chemistry (6th Edition). John Wiley & Sons, Hobken, NJ.
58
- Example: A set of data was obtained from the measurement of a series of
standard solutions of metal ion A.
Concentration of
metal ion A in the
standard (ppm) Absorbance
0 0.20
1 0.35
2 0.50
3 0.85
4 1.00
An Uncorrected Calibration Curve Constructed Based on the Data Obtained
- The R2 value is known as the coefficient of determination, which ranges

from 0 to 1. An R2 value of 1 means that all the data points fitted nicely with
the “best” straight line.
59
- The most simplest way to determine the value of k in Equation 10 is by a single-point
standardization.
- A single standard containing a known concentration of analyte, Cstandard and its signal,
Sstandard is measured.
- The value of k is calculated by referring to Equation 11,
k = Sstandard /Cstandard Equation 12
- Single-point standardization, however, is the least desirable way to standardize a

method.
- When using a single standard, all experimental errors, both determinate and
indeterminate, are carried over into the calculated value of k. Any uncertainty in the value
of k increases the uncertainty in the analyte’s concentration.
References:
60
- The preferred approach for a method standardization is to prepare a series of standards,
each containing the analyte at a different concentration. Standards are chosen such that
they bracket the expected range for the analyte’s concentration. This method is known
as the multiple-point standardization.
- A multiple-point standardization should use at least three standards, although more are
preferable.
- Once the signals of all the standards Sstandard are obtained, a calibration curve can be
constructed.
- The k value for the multiple-point standardization method can be obtained from the
slope of the calibration curve.
References:
61
- Almost all analytical methods require some type of calibration with chemical standards.
- Gravimetric methods and some coulometric methods are among the few absolute
methods that do not rely on calibration with chemical standards.
- The reagents used as standards are divided into primary reagents and secondary
reagents.
> A primary reagent can be used to prepare a standard containing an accurately known
amount of analyte. For example, an accurately weighed sample of 0.1250g K2Cr2O7
contains exactly 4.249 x 10-4 mol of K2Cr2O7. If this is placed in a 250-mL volumetric
flask and diluted to volume, the concentration of the resulting solution is exactly 1.700
x10-3M.
>Reagents that do not meet the criteria of the primary reagents are called secondary
reagents.
References:
- Skoog, C. D., West, D. M., Holler, F. J., & Crouch, S. R. (2004). Fundamentals of Analytical Chemistry (8th
Edition), John Wiley & Sons, Hoboken , NJ.
- Substances that are to be used as primary standards must meet some
special requirements with respect to purity, etc.; these are enumerated below:
> It must be 100% pure, or at least its purity must be known.

> If it is impure, the impurity must be inert.
> It should be stable at drying oven temperatures.
> It should be not hygroscopic – it should not absorb water when
exposed to laboratory air.
> The reaction in which takes part must be quantitative and preferably fast.
> A high formula weight is desirable, so that the number of significant
figures in the calculated results is not diminished.
References:
- Kenkel, J. (2003). Analytical Chemistry for Technicians (3 Edition). CRC Press, Boca Raton.
63
- Examples of the Commonly Used
Calibration methods
S standard
> External Standard Calibration (a)
> Internal Standard Calibration (b) Cstandard
> Standard Addition (c) (a)

Sanalyte/Sinternal standard
S standard
Canalyte/Cinternal standard Cstandard
(c)
(b) 64
- External Standard Calibration
> Measure the signal of a single standard (single-point standardization) or the signals of a
series of standard solutions (multiple-point standardization).
> The standards are identified as external standards because they are prepared and analyzed
separately from the samples.
> For single external standard, the concentration of the analyte in an unknown sample (Csample)
can be determined by rearranging Equation 11.
Csample = Ssample / k Equation 13
where Ssample is the signal given by the analyte in the unknown sample. The k value can be
calculated by referring to signal and concentration of a standard solution and calculated using
Equation 12.
> For multiple external standards calibration, the calibration curve and the equation for the line
can be used to determine the concentration of the analyte in the unknown sample based on its
signals.
65
External Calibration (Multiple standards) - Example
Concentration of Cu Signal Corrected signal

standard solutions (ppm) (Absorbance) (Corrected absorbance#)
0.0 0.002 0.000*

(blank)
1.0 0.078 0.076
2.0 0.163 0.161
4.0 0.297 0.295
6.0 0.464 0.462
8.0 0.600 0.598
* Zero absorbance is defined with a blank

# Corrected absorbance = (absorbance of sample) – (absorbance of blank)
66
External Calibration (Multiple standards) – Example (continued)
0.7
0.6
0.5
0.4 Standard calibration
Abs
0.3 curve
0.2
0.1
0
0 1 2 3 4 5 6 7 8 9
Conc (ppm)
0.7
0.6
0.5
0.4 Get the best fit
Abs
0.3
straight line
0.2 y = 0.075x
0.1 R2=0.9991
0
0 1 2 3 4 5 6 7 8 9
Conc (ppm)
67
External Calibration (Multiple standards) – Example (Continued)
-If a sample solution give an absorbance value of 0.4, the concentration of analyte
in the sample can then be determined from the calibration curve and the equation
for the line.
0.7
0.6
0.5
0.4
Abs
0.3
0.2 y = 0.075x
0.1 R2=0.9991
0
0 1 2 3 4 5 6 7 8 9
Conc (ppm)
5.3 ppm
-The concentration of the bulk can be determined by multiplying the concentration
of the analyte in the sample with the dilution factor.
68
- Internal Standard Calibration
> An internal standard refers to a reference species, chemically and physically similar to the
analyte, that is added to samples, standards, and blanks.
> The internal standard should be chemically inert to the sample components and must be
available in high purity.
> The response from the analyte is compared to that of internal standard. The response
factor F can be calculated using the folllowing equation. It can also be obtained from a
calibration curve and line equation (see example in the next slide).
Equation 14
Where Sanalyte and Sis are the signals for the analyte and internal standard while Canalyte and
Cis are the concentrations of analyte and internal standard.
69
- Internal Standard Calibration (continued)
> The concentration of the analyte in an unknown sample can be determined once the F
value is known. It can also be obtained from a calibration curve.
> The calibration curve for internal standard calibration is a plot of the ratio of the response
analyte to that of the internal standard (Sanalyte/Sis) versus the concentration of analyte
(Canalyte ) OR the ratio of the concentrations of analyte and the internal standard (Canalyte /Cis).
Sanalyte/Sis
Sanalyte/Sis
Canalyte Canalyte/Cis
Chemistry (8th Edition), John Wiley & Sons, Hoboken , NJ. 70
An Example of Internal Standard Calibration – Analysis of Cocaine Content using
Gas Chromatography (GC).
Standards GC Calibration Curve for Cocaine with Internal Standard
• Each contains fixed mass of internal Cocaine Int. Std.

standard, various masses of standards Standard mg/mL mg/mL cx/cis Ax Aix Ax/Ais
1 2.50 5.00 0.500 120 600 0.200
analyte 2 5.00 5.00 1.000 241 601 0.401
• Plot AX/Ais vs cx/cis (AX and Ais are the peak 3 10.00 5.00 2.000 480 600 0.800
4 25.00 5.00 5.000 1198 600 1.997
areas of the analyte and internal standard)
• Calibration curve shows linear response.
Cocaine with Interal Standard
Slope = response factor 1.0 microliter injections
2.500
y = 0.3991x + 0.0013
2.000
1.500 F
Ax/Ais
Unknown 1.000
• Add known amount of internal standard
0.500
cis is known
• Measure Ax/Ais 0.000
• Determine cx/cis from calibration data, and 0.000 1.000 2.000 3.000 4.000 5.000 6.000
cx/cis
calculate cx
c 
c x  cis  x 
 c is 
Why need to use internal standard method?
• The internal standard method can compensate for certain types of errors if
these influence both the analyte and the reference species to the same
proportional extent.
• For example, if temperature influences both the analyte and reference
species to the same extent, taking the ratio can compensate for variations
in temperature.
•For compensation to occur, the reference species is chosen to have very
similar chemical and physical properties to the analyte.
72
- Standard Addition Method
> Generally, the analyte is not isolated from other components in the sample.
> The matrix is everything in the sample except the analyte.
> Sometimes certain components may interfere with the analysis by either enhancing or
suppressing the analytical signal  matrix effect
> However, the extent to which the signal is affected is difficult to measure.
> There is a useful technique that often can make it possible to work in the presence of a
matrix interference without eliminating the interference itself, and still make an accurate
determination of analyte concentration.
> Standard Addition: add a small volume of concentrated standard solution to a known
volume of the unknown.
Assumptions:
Aliquots of a standard are added to portions of the sample, thereby
allowing any interference present in the sample to also affect the
standard similarly.
73
- Standard Addition Method (continued)
How is this done in practice?
1. Take equal aliquots of the sample

2. Nothing is added to the first
aliquot
3. A measured amount of standard
is added to the second; and a
larger measured amount is added
to the third and so forth
4. Finally, all the portions are diluted
to the same volume so that the
final concentration of the original
sample constituents are the same
in each case
74
> The solutions in the above flasks all have the same amount of the matrix.
> Only the amount of added standard differs, and then by a known amount.
> Usually add a quantity of standard solution such that the signal is increased
by about 1.5 to 3 times that of the original sample.
(b)
(a)
Standard Calibration Method Standard Addition Method

Solid line (a) – passing through the If no interferences were present in the
origin  a typical calibration line for a sample, a plot of measured absorbances
set of standard solutions versus the concentrations of added standards
(b) would be parallel to the standard
calibration (a) 75
> Recall Equation 11, Ssample = k.Canalyte

> For standard addition, the standards are added into the sample solution,
therefore both concentrations of the sample and the added standards
contributed to the signal observed. Equation 11 is modified to give
Sss = k.(Cstandard + Canalyte) Equation 15
Cstandard = Concentration of the added standard

Canalyte = Concentration of the analyte solution
SSS = Signal of the sample plus standard
76
Absorbance (Ass)
> From Equation 15, Sss=k(Cstandard +Canalyte), a plot of Sss (Ass in this
example) versus Cstandard is a straight line which can be extrapolated
to Ass=0 to give Canalyte = ─ Cstandard. The concentration responsible
for the absorbance of the unknown sample can then be obtained.
> An accurate determination has been made using a standard addition
77
method by calibrating in the presence of the interference!

Topic 7: Sampling, Standardization and Calibration

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Topic 7

Sampling, Standardization and

What is Qualitative & Quantitative analyses?

Qualitative analyses : To identify chemical species

Quantitative analyses : To determine the amount of a constituent

To distinguish suitable analyses methods is based on the size of sample and

 Gravimetric Methods determine the mass of the analyte or some compound

 Volumetric Methods measure the volume of a solution containing sufficient reagent

 Electroanalytical Methods involve the measurement of such electrical properties as

 Spectroscopic Methods are based on the measurement of the interaction between

 Miscellaneous Methods include the measurement of such quantities as mass-to-

Obtain a Prepare a Define

0.0001 0.001 0.01 0.1

- Analysis of a 1g sample of soil for a suspected pollutant would be called a macro

Type of Constituent Major

International Organization for ASTM International

AOAC International American Public Health Association

- Involves obtaining a small mass of a material whose composition accurately

- Steps in obtaining a laboratory sample:

Reduce the gross

- The population refers the bulk materials being sampled.

- The collection of sampling units or increments are called gross samples.

The collection of coins Five coins were collected

(say 1000 coins) (Each coin is known as a SAMPLING UNIT or

POPULATION SAMPLING INCREMENT)

The coins sample was reduced in size

ii. To obtain a variance that is an unbiased estimate of the population variance so

- Both goals requires a random sample, a sample chosen by applying

Taken from http://www.pitt.edu/~super1/lecture/lec0411/022.htm

Division of the flattened cone

Removal of alternate quarters to define Further division of the flattened cone

H. G. Brittain. (2002). Pharmaceutical Technology, Page 67 18

Soverall2 = Ssampling2 + Smethod2 Equation 1

- Basically, the gross sample weight is determined by:

- To obtain a truly representative gross sample, a certain number N of particles must be

d = average density of particles (differs from the densities of particles A, dA

P = overall average percentage

σr = Sampling relative standard deviation

Mass of cereal (g) Percentage ash (w/w)

Reference: Harvey, D. (2000). Modern Analytical Chemistry. McGraw-Hill Publishing, Singapore.

Reference: Harvey, D. (2000). Modern Analytical Chemistry. McGraw-Hill Publishing, Singapore.

Reference: Harvey, D. (2000). Modern Analytical Chemistry. McGraw-Hill Publishing, Singapore.

A) Homogeneous solutions of Liquids and Gases.

Weighted Beaker Sampler can be

Zone Sampler can be

Stainless Steel & Brass Weights

The CS Restricted sampler shown can be used to obtain: RVP Sample,

A portable water testing equipment A portable Gas Chromatography-

> Several methods of collecting gases samples:

Randomly collect N Reduce particle size of

Store the laboratory Is this sample of a Randomly collect N

Remove portion of the laboratory

- For homogenous solids, the gross sample may weigh

- The laboratory sample should have the same number

- The process of preparation of a laboratory sample

- The number of laboratory samples N required to obtain a

where t is the value obtained from the t-table at the given

The determination of copper in a sea water sample gave a mean

The collection of coins Five coins were collected

The lead in the coins was extracted by

THREE REPLICATE SAMPLES

- The purpose of performing chemical analyses on replicate samples is to

where e2 is expressed as absolute uncertainties or as relative uncertainties.